Kinetic characterization using Biacore™ cap-tag capture kit

Elin Sivertsson, Scientist
Claire Shepherd, Global Product Manager Biacore Consumables

Abstract

We describe how we used Biacore™ surface plasmon resonance (SPR) system, Biacore cap-tag capture kit, and Biacore regeneration kit CAP to rapidly characterize the binding of a nanobody to its protein target, without any prior knowledge of their interaction kinetics.

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) causes the respiratory disease coronavirus disease 2019 (COVID-19). To gain entry to a host cell, the receptor binding domain (RBD) of the SARS-CoV-2 spike protein binds to angiotensin-converting enzyme 2 (ACE2) on the host cell surface (1).

Nanobodies, or VHH single-domain antibodies, are derived from heavy-chain only antibodies found in camelids. They have gained increasing interest from researchers due to their potential use in research, diagnostic, and therapeutic applications. Advantages include their small size, high chemical and thermal stability, and ease of production (2).

We aimed to determine the binding kinetics of a commercially available nanobody binding to RBD (here called anti-RBD nanobody). To characterize this interaction, we used Biacore cap-tag capture. This approach provides multiple advantages:

1. Simple protein capture regardless of tag.
2. No development of attachment or regeneration conditions.
3. Reusable and cost-effective.

Developing an assay using amine coupling can be an iterative process. Steps such as pH scouting to find a suitable coupling solution for effective ligand preconcentration and regeneration scouting to find a solution that removes the analyte without damaging the ligand may need to be repeated multiple times before satisfactory conditions are found (Fig 1). This can increase both the time spent on the instrument and the cost of materials, as multiple sensor chips may need to be used and discarded. Additionally, some ligands are unstable and may not tolerate the low pH used during amine coupling or the repeated exposure to regeneration solutions.

Biacore cap-tag capture kit allows you to attach your protein of interest to Series S Sensor Chip CAP by first conjugating a short oligonucleotide tag to the protein, which then binds to the complementary oligo on the sensor chip. The produced ligand conjugate is used in reversible capture for protein interaction analysis in Biacore systems. Regeneration of the surface after each analysis cycle removes the ligand conjugate and any bound analyte. Fresh ligand conjugate is attached to the surface for each cycle. The sensor chip can be reused at least 150 times per flow cell (Fig 1).

Fig 1. Workflow of an amine-coupling based assay compared to a Biacore cap-tag capture assay.

* Multiple cycles can be run on the same surface. However, the flow cell cannot be reused with a different ligand.

Results and discussion

Preparation of ligand conjugate

We prepared the RBD ligand conjugate according to Biacore cap-tag capture kit instructions for use (IFU). For details, see Materials and methods. Note that Biacore cap-tag conjugation adds an additional 7 kDa to the original molecular weight of the ligand protein used. The molecular weight of the RBD ligand conjugate is thus 25 kDa + 7 kDa = 32 kDa.

Capture level scouting

We wanted an R_max of ~ 5-10 RU in our kinetics assay. Each RBD molecule can bind one anti-RBD nanobody molecule. The molecular weight of the anti-RBD nanobody is approximately 15 kDa. We used equation (1) in Materials and methods:

$$R_{\mathrm{RBD} \mathrm{ligand} \mathrm{conjugate}}=\frac{10 \mathrm{RU}}{1}\times \frac{32 \mathrm{kDa}}{15 \mathrm{kDa}}\approx 21 \mathrm{RU}$$

To establish how much we should dilute the RBD ligand conjugate to reach a capture level of 21 RU, we performed capture level scouting at a range of dilution factors. You can see the sensorgrams from the capture level scouting in Figure 2. We looked at the plot of capture level versus dilution factor and found that to reach a capture level of ~ 20 RU, we should dilute the RBD ligand conjugate 3000 times (Fig 3). It was helpful to use logarithmic axes in the plot to more easily visualize the relationship between capture level and dilution factor.

Fig 2. Capture level scouting sensorgrams. A dilution series of RBD ligand conjugate was injected over Series S Sensor Chip CAP. The capture baseline and capture level report points are shown in black.

(A)

(B)

Fig 3. Plot of capture level versus dilution factor for the RBD ligand conjugate. The (A) plot on linear axes. The (B) plot on logarithmic axes. We have added a dashed line that connects the data points as a guide to the eye.

Biacore Single-Cycle Kinetics (SCK) analysis

Initial run

Next, we performed Biacore Single-Cycle Kinetics (SCK) analysis to characterize the binding between RBD and the anti-RBD nanobody. Running the assay in the SCK format reduces the amount of RBD ligand conjugate needed per run. As we had no prior knowledge of the affinity or kinetics of the interaction between RBD and the anti-RBD nanobody, we used the predefined settings in the provided analysis method (see Materials and methods). These predefined settings cover a wide analyte concentration range and can be used to give a first indication (or, in the best case, a determination) of the kinetic and affinity properties. We diluted the RBD ligand conjugate 3000 times, based on the results of the capture level scouting.

Figure 4 shows the results of the initial SCK run, analysed in Biacore Insight evaluation software. We noted the following:

1. The RBD ligand conjugate dilution factor is suitable. The maximum analyte binding response is around 10 RU, which is what we wanted. The capture level was 23.2 RU and 23.5 RU in the blank and sample cycles, respectively.
2. The anti-RBD nanobody concentration is too high. The binding is saturated already after the third injection (60 nM anti-RBD nanobody). No useful data is obtained from the fourth and fifth injection.
3. The dissociation time is too short. The dissociation of the anti-RBD nanobody from the RBD ligand conjugate is very slow. During the 10 minute dissociation phase, hardly any signal is lost. For reliable evaluation of the dissociation rate constant, a dissociation time that allows a drop in response of about 10% or more of the starting value is recommended.

Fig 4. Sensorgram (reference subtracted and blank subtracted, orange) from the initial SCK run, fitted to a 1:1 binding model (black). RBD ligand conjugate was captured on Series S Sensor Chip CAP and a concentration series of anti-RBD nanobody was injected. The bars show the SCK injections of the indicated anti-RBD nanobody concentrations.

While the predefined parameters used here are not optimal for this interaction, we can still get an indication of the interaction kinetics by fitting a 1:1 binding model to the data. The fitted parameters are k_a = 6.92 x 10⁵ M^-1s^-1 and k_d = 3.35 x 10^-7 s^-1.

Simulation

We put in the estimated kinetic parameters from the initial SCK run in the simulation tool Biacore Simul8 (Fig 5). We then tested the analyte concentration in the simulation. We knew from the initial run that an analyte concentration above 60 nM was too high (see the fourth and fifth injections in Fig 4). In the simulation, five injections with a top concentration of 30 nM and a dilution factor of 3 (i.e., 0.37, 1.11, 3.33, 10, 30 nM) gave a good result. The first injection gives a measurable response, and the final injection approaches saturation.

In the simulation we also increased the SCK contact time from 120 s to 180 s, to achieve more curvature in the later injections. We also increased the dissociation time to get a better chance of determining the slow off-rate.

Fig 5. Screenshot of simulations of SCK sensorgrams in Biacore Simul8, based on estimated kinetic parameters from the initial run.

Refined SCK run

We then set up a refined SCK run, using the concentration range and association time from the simulation. We increased the dissociation time to the maximum value (10 000 s). We also included three SCK blank cycles at the start of the run. This can help stabilize the system which is extra important when working with samples with slow dissociation. We ran SCK sample cycles in triplicate, interspersed with blank cycles (see Materials and methods for details).

Figure 6 and Table 1 show the result from the refined SCK run, analyzed in Biacore Insight evaluation software.

(A)

(B)

(C)

Fig 6. Results of the refined SCK run. (A): Sensorgram (reference subtracted and blank subtracted, orange) fitted to a 1:1 binding model (black). The inset shows the first 1500 s of the sensorgram (framed in dashed line). (B): Residuals plot for the kinetics fit. (C): Screen shot of the quality control tab from Biacore Insight evaluation software.

Table 1. Fitting results and statistical parameters for the refined SCK run.

ka (1/Ms)	T(ka)	kd (1/s)	T(kd)	Kd (M)	Rmax (RU)	tc	U-value	Kinetics Chi² (RU²)
7.06 x 105	3.33 x 103	1.43 x 10-6	2.08 x 102	2.03 x 10-12	7.2	1.43 x 1010	7	2.07 x 10-3

We note the following:

1. The anti-RBD nanobody concentration range is appropriate. As in the simulation based on the initial run, the first injection gives a measurable response, the later injections have sufficient curvature and the final injection approaches saturation (Fig 6A).
2. The 1:1 model is suitable for this interaction. The residuals (Fig 6B) are close to zero with no systematic deviations, indicating a good fit. The fit results are k_a = 7.06 x 10⁵ M^-1s^-1 and k_d = 1.43 x 10^-6 s^-1. For detailed fitting results, see Table 1.
3. The activity of the ligand conjugate on the surface is ~ 80%. The capture level of the RBD ligand conjugate was 19.1 RU, from which we can calculate a theoretical R_max of 9.0 RU (see Equation (1) in Materials and methods). The fitted R_max was 7.2 RU, that is ~ 80% of the theoretical value.
4. The dissociation is very slow and k_d is difficult to determine with certainty. The orange symbol in the quality control panel in Biacore Insight evaluation software (Fig 6C) warns that the dissociation rate constant k_d is approaching the limits that can be measured by the instrument.

We discuss the last two points in more detail below.

R_max

There could be several reasons for the observed R_max value being lower than the theoretical value:

1. The equation assumes that the ligand on the surface is 100% active. Note that in practice, the activity of biological samples is usually less than 100%. This applies to ligand attachment in general, not just to capture using Biacore cap-tag capture kit.
2. A certain amount of unconjugated Biacore cap-tag may be left in the conjugate preparation after the final purification. The unconjugated Biacore cap-tag will occupy a fraction of the binding sites on Series S Sensor Chip CAP, thereby contributing to the capture response level but not to the analyte binding response level. This causes the observed R_max to be reduced compared to the theoretical R_max.

Slow dissociation

Generally, a dissociation time that allows a drop in response of about 10% or more of the starting value is recommended. Even though we used the maximum dissociation time allowed by the system here (10 000 s), we did not achieve a 10% response drop but are still able to measure a dissociation rate constant for an interaction approaching the limits of Biacore 1K+ SPR system using Biacore cap-tag capture kit. Note that an interaction with a k_d of 10^-6 s^-1 will take approximately 29 hours for a 10% response drop (3). Very slow dissociations have been successfully characterized using a SPR 'chaser assay' (4).

Reagent consumption and run time

Biacore cap-tag capture kit allows you to analyze multiple proteins using the same kit. Series S Sensor Chip CAP can be reused at least 150 times per flow cell. On a Biacore 1K+ system you could run 450 cycles using the set-up presented here (three flow cell pairs). Normally, the yield of ligand conjugate is high, meaning that it must be diluted many times to get capture levels suitable for kinetic analysis. From 50 µL RBD ligand conjugate diluted 3000 times, it would be possible to run over 3400 cycles with the set-up used in this experiment. It would also be possible to run over 425 cycles from the regeneration solution provided in one unit of Biacore regeneration kit CAP. Therefore, the assay set up described here would be suitable for kinetic characterization or screening of a nanobody library against RBD. Alternatively, Series S Sensor Chip CAP can be stored and reused for multiple projects to decrease consumables cost.

The run time to perform capture level scouting was 2 h 6 min, and to perform the initial SCK run 2 h 19 min. Note that the fitted k_a was very similar between the initial run and the refined run. The fitted k_d was four times larger in the refined run. This is still close to the limits that the instrument can measure, meaning there is larger uncertainty here.

Conclusions

1. We characterized the kinetics of binding of a nanobody (anti-RBD nanobody) to its protein target (RBD) using a Biacore cap-tag approach on Biacore 1K+ SPR system.
2. No time or material was spent on developing attachment or regeneration conditions saving an estimated half day of instrument time, or even several days and multiple chips.
3. The instrument time used to get an initial estimation of the kinetics was less than 5 h.
4. We found that the interaction had high affinity with very slow dissociation.
5. The set-up presented here allows for reuse of the sensor chip in 450 cycles (150 cycles per flow cell), 425 cycles of regeneration and 3400 cycles of ligand conjugate capture. This corresponds to around 400 yes/no binding interactions or 200 kinetic interactions to be measured using one sensor chip in a single needle system, making it a cost-effective choice.

Explore new horizons with one capture approach

Learn more about Biacore cap-tag capture kit on our website. You'll find a product data file and links to our how-to video.

Curious to try it out? Sign up here to connect with our sales or application specialist for more information and to arrange a trial.

References

1. Cui W, Duan Y, Gao Y, Wang W, Yang H. Structural review of SARS-CoV-2 antiviral targets. Structure. 2024;32(9):1301-1321. doi: 10.1016/j.str.2024.08.005
2. Alexander E, Leong KW. Discovery of nanobodies: a comprehensive review of their applications and potential over the past five years. J Nanobiotechnology. 2024;22(1):661. doi: 10.1186/s12951-024-02900-y
3. Application guide: Kinetics and affinity measurements with Biacore systems. Cytiva, CY12854-21Jan21-HB; 2021.
4. Quinn JG, Pitts KE, Steffek M, Mulvihill MM. Determination of Affinity and Residence Time of Potent Drug-Target Complexes by Label-free Biosensing. J. Med. Chem. 2018; 61(12):5154-5161. doi: 10.1021/acs.jmedchem.7b01829.

Materials and methods

Conjugation of RBD

Preparations of conjugation working solutions

Biacore conjugation buffer, Biacore ligand activator and Biacore cap-tag is provided in Biacore cap-tag conjugation kit. We prepared 1 x Biacore conjugation buffer by dilution of the 5 x concentrated stock. We dissolved Biacore ligand activator and Biacore cap-tag according to the instructions for use of Biacore cap-tag capture kit.

Dilution and buffer exchange of RBD

We diluted RBD (prepared in house, MW 25 kDa, stock concentration 2.5 mg/mL) to 1 mg/mL in 1 x Biacore conjugation buffer. We buffer exchanged the diluted RBD on an Amersham™ MicroSpin™ G-50 column (pre-washed four times in 1 x Biacore conjugation buffer, according to Biacore cap-tag capture kit instruction for use).

Activation of RBD

The instruction for use document states that $V_{\mathrm{Activator}}(\text{μL})=\frac{64.7}{{\mathrm{MW}}_{\mathrm{ligand}}\left(\mathrm{kDa}\right)}$

We therefore added 64.7/25=2.59 µL of Biacore ligand activator directly to the desalted RBD eluate, before mixing well and incubating for one hour at 25 °C (protected from light). Immediately after the incubation, 50 µL of the reaction mixture was purified on an Amersham MicroSpin G-50 column (pre-washed four times in 1 x Biacore conjugation buffer).

Conjugation of Biacore cap-tag to activated RBD

The instruction for use document states that $V_{\mathrm{Cap}-\mathrm{tag}}(\text{μL})=\frac{95.2}{{\mathrm{MW}}_{\mathrm{ligand}}\left(\mathrm{kDa}\right)}$

We therefore added 95.2/25=3.81 µL of Biacore cap-tag directly to the purified activated RBD, before mixing well and incubating for one hour at 25 °C. Immediately after the incubation, 50 µL of the reaction mixture was purified on a Amersham MicroSpin G-50 column (pre-washed four times in 1 x Biacore conjugation buffer). The eluted ligand conjugate was stored at -20 °C for later analysis.

Biacore interaction analysis

General preparations

We used a Biacore 1K+ SPR instrument. We docked a Biacore Series S Sensor Chip CAP and allowed it to rehydrate by leaving it on standby in HEPES buffered saline containing EDTA and polysorbate (HBS-EP+) overnight.

We prepared regeneration solution from Regeneration stock 1 and 2 provided in Biacore regeneration kit CAP.

Capture level scouting

We calculated a suitable capture level using the following formula:

$R_{\mathrm{ligand }\mathrm{conjugate}}=\frac{R_{\max }}{n}\times \frac{{\mathrm{MW}}_{\mathrm{ligand }\mathrm{conjugate}}}{{\mathrm{MW}}_{\mathrm{analyte}}}$ (1)

Term	Meaning
Rligand conjugate	Ligand conjugate capture level (RU)
Rmax	Theoretical maximum analyte binding capacity (RU)
MW	Molecular weight
N	Interaction stoichiometry (analyte/ligand)

We prepared a serial dilution of the RBD ligand conjugate in HBS-EP+ (two-fold dilutions, ranging from 40 times dilution to 5120 times dilution). We performed capture level scouting using the predefined Biacore Insight control software run method. We have listed the run parameters in Table 2. We used an analysis temperature of 25°C.

Table 2. Run parameters for capture level scouting.

Cycle	Purpose	Command	Solution	Concentration	Contact time	Flow rate	Flow cell
1	Conditioning	Regeneration	Regeneration solution	N/A	Three 60 s pulses	10 µL/min	3 and 4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A
2	Startup	Capture	RBD ligand conjugate	5120 times diluted	300 s	2 µL/min	4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A
		Regeneration	Regeneration solution	N/A	120 s	10 µL/min	3 and 4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A
3-10	Analysis	Capture	RBD ligand conjugate	5120 to 40 times diluted*	300 s	2 µL/min	4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A
		Regeneration	Regeneration solution	N/A	120 s	10 µL/min	3 and 4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A

*Different concentrations in each cycle, from low to high.

Biacore Single-Cycle Kinetics (SCK) analysis

We diluted the RBD ligand conjugate 3000 times (as established in the capture level scouting) in HBS-EP+. We prepared five concentrations of SARS-CoV-2 Spike RBD Llamabody VHH His-tag Antibody (R&D Systems, here called anti-RBD nanobody) in HBS-EP+. We have listed the run parameters of the initial SCK run in Table 3. We used an analysis temperature of 25 °C.

Table 3. Run parameter for Biacore Single-Cycle Kinetics (SCK) analysis.

Cycle	Purpose	Command	Solution	Concentration	Contact time	Dissociation time	Flow rate	Flow cell
1	Conditioning	Regeneration	Regeneration solution	N/A	Three 60 s pulses	N/A	10 µL/min	3 and 4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A	N/A
2-4	Startup	Capture	RBD ligand conjugate	3000 times diluted	300 s	N/A	2 µL/min	4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A	N/A
		Analyte (High performance)	HBS-EP+	N/A	120 s	60 s	30 µL/min	3 and 4
		Regeneration	Regeneration solution	N/A	120 s	N/A	10 µL/min	3 and 4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A	N/A
5	Analysis (blank)	Capture	RBD ligand conjugate	3000 times diluted	300 s	N/A	2 µL/min	4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A	N/A
		Single cycle kinetics	Anti-RBD nanobody	0 nM	120 s	600 s	30 µL/min	3 and 4
		Regeneration	Regeneration solution	N/A	120 s	N/A	10 µL/min	3 and 4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A	N/A
6	Analysis (sample)	Capture	RBD ligand conjugate	3000 times diluted	300 s	N/A	2 µL/min	4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A	N/A
		Single cycle kinetics	Anti-RBD nanobody	2.4, 12, 60, 300, 1500 nM	120 s	600 s	30 µL/min	3 and 4
		Regeneration	Regeneration solution	N/A	120 s	N/A	10 µL/min	3 and 4
		Wash	HBS-EP+	N/A	N/A	N/A	N/A	N/A

In the refined run, we increased the SCK contact time to 180 s and the SCK dissociation time to 10 000 s. We also reduced the SCK analyte concentration (0.37, 1.11, 3.33, 10, 30 nM). We included additional analysis cycles so that the run had a total of eight SCK cycles in the following order: 3 blank cycles, 1 sample cycle, 1 blank cycle, 2 sample cycles, 1 blank cycle.

Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an affiliate doing business as Cytiva.

Amersham, Biacore, Biacore Single Cycle Kinetics (SCK), and MicroSpin are trademarks.

NANOBODY and NANOBODIES are trademarks of Ablynx N.V. Any other third-party trademarks are the property of their respective owners.

EXP-020572

© 2025 Cytiva

CY48560-12Jun25-CS